Biochemical transformation of solid carbonaceous material

ABSTRACT

A method of biochemically transforming macromolecular compounds found in solid carbonaceous materials, such as coal is provided. The preparation of new microorganisms, metabolically weaned through challenge growth processes to biochemically transform solid carbonaceous materials at extreme temperatures, pressures, pH, salt and toxic metal concentrations is also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional of application Ser. No. 08/344,126 now U.S. Pat.No. 5,885,825, filed Nov. 23, 1994 and a continuation-in-part ofapplication Ser. No. 08/169,417 filed Dec. 20, 1993 now U.S. Pat. No.5,492,828, which is a divisional application of application Ser. No.07/905,391 filed Jun. 29, 1992 now U.S. Pat. No. 5,297,625 issued Mar.29, 1994 in the names of Eugene T. Premuzic and Mow Lin entitled“Biochemically Enhanced Oil Recovery and Oil Treatment”, which is acontinuation-in-part of application Ser. No. 07/571,917 filed Aug. 24,1990 and now abandoned.

This invention was made with government support under contract numberDE-AC02-76CH00016, between the U.S. Department of Energy and AssociatedUniversities, Inc. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

The present invention relates to biochemical transformation of solidcarbonaceous material by a process of depolymerizing, desulfurizing anddemineralizing using modified strains of thermophilic bacteria. Morespecifically, the present invention provides a process of treating anaqueous slurry of solid carbonaceous material, such as coal, with aculture of thermophilic bacteria which have been modified throughchallenge growth processes to be suitable for microbially enhanced oilrecovery. Some of these bacteria were further adapted to grow on coalunder challenged growth conditions and then utilized for biochemicaltransformation of solid carbonaceous material.

Coal is a solid carbonaceous material which is one of the most abundantfossil energy resources accessible to mankind. In terms of environmentalprotection, coal combustion is associated with significant problems,such as sulfur and nitrogen oxides emission, and the production of toxicmetal containing ash. However, coal can be treated prior to combustionin a manner which reduces sulfur and ash contents. The treated productbecomes a more suitable feedstock for combustion, liquefaction andgasification processes.

Sulfur appears in coal in three basic forms: as sulfates, pyrites andorganic sulfur. Of the three forms, sulfates are the least significant,comprising less than 0.5 weight percent of coal. Pyritic and organicsulfur, however, may each constitute as much as 3.5 weight percent ofthe coal or from 40% weight to 60% weight of the total sulfur content,respectively. Thus, it is apparent that removal of an effective portionof inorganic and organic sulfur content prior to coal combustion couldsubstantially reduce the emission of sulfur oxides into the atmosphere.

By organic sulfur is meant sulfur which is chemically bound within thecoal matrix. Organic sulfur is present in four major forms. These aremercaptans or thiols, sulfides, disulfides and aromatic ring sulfur asexemplified by the thiophene system.

In the past, attempts have been made to alleviate the environmentalproblems associated with the combustion of coal. A number of processesfor the treatment of coal ranging from ash and pyrite reduction toorganic sulfur removal and coal liquefaction have been reported. Theseprocesses include numerous physical and mechanical techniques such asheavy media separation, elective agglomeration, flotation, jigging,magnetic, separation, leaching and hydrosulfurization. Problemsassociated with these developing technologies include reproductivity,slow kinetics and scale-up difficulties.

The removal of inorganic sulfur from pyrite is relatively easilyaccomplished by treatment with dilute nitric acid. The removal oforganic sulfur, however, has not met with the same success. In recentyears in order to obtain cleaner coal, coal has been subjected tobiochemical processes wherein inorganic sulfur and minerals are removedthrough the action of several microorganisms belonging to theThiobacillus, Sulfolobus and Pseudomonas Clostridium species. Forexample, U.S. Pat. No. 4,632,906 to Kopacz discloses a process forbiodesulfurization by contacting carbonaceous material with BacillusSulfasportate of ATCC No. 39909. A mutant of the family Bacillaceae,this microorganism grows in a range between 15° C. to 30° C. and is nota thermophile. The background of the invention of the '906 disclosuredescribes Sulfolobus Acidocaldarius, a thermophilic sulfur and ironoxidizing microorganism which is a facultative autotroph. This organismhas been used for removal of primarily pyritic sulfur from coal.

U.S. Pat. No. 4,206,288 to Detz et al. discloses a process for removalof pyritic sulfur from coal by microbial desulfurization of coal slurrywith iron and sulfur oxidizing microorganisms selected from theThiobacillus ferooxidans species. The microorganisms described in the'288 disclosure must be kept in a range of about 10° C. to 35° C. andare not thermophilic as required by the process of the presentinvention. Thus, they cannot be used at elevated temperatures.

U.S. Pat. No. 4,562,156 to Isbister et al. describes a mutantmicroorganism Pseudomonas sp. CB1 (ATCC 39381) used in the removal oforganic sulfur compounds from carbonaceous materials including coal. Inaddition, the '156 disclosure describes other genera of microorganismssuch as Arthrobacter and Acinetobacter used for microbiologicaltreatment of petroleum and coal. These microorganisms are notthermophiles because they cannot grow at 41° C.

Generally, the microorganisms described in the above disclosures are notresistant to challenged environments, namely those including elevatedtemperatures, pressures, low pH, high levels of salinity and toxicmetals. Frequently, it has been found more economical to process coalunder elevated temperatures and pressures. Moreover, many types of coalhave high toxic metal content which can kill many strains of bacteriaeven those which have been previously environmentally challenged.

Accordingly, there is a need in the art of biochemical transformation ofsolid carbonaceous materials for microorganisms which can depolymerize,desulfurize and/or demineralize solid carbonaceous material such as coalunder environmentally challenged conditions.

It is therefore, an object of the present invention to provide modifiedthermophilic bacteria which are useful for the biochemicaltransformation of coals in a challenged environment.

SUMMARY OF THE INVENTION

The present invention, which addresses the needs of the prior art,provides a method of biochemically transforming complex macromolecularcompounds found in solid carbonaceous materials, such as coal. Morespecifically, coal is treated with a biological treatment mediumincluding strains of bacteria which have been metabolically weaned underchallenged conditions to metabolize the complex macromolecular compoundsfound in coal thus providing a solid carbonaceous material which hasbeen depolymerized, desulfurized and/or demineralized.

The biological treatment medium is prepared by nutritionally stressingthermophilic microorganisms to metabolize initially a non-solidcarbonaceous material, such as crude oil at a desired temperature,pressure, pH and salinity.

The strains which survive in the presence of crude oil are thensubjected to further challenge by selection under more extremeconditions. The selection process proceeds by the removal of the moreeasily metabolizable carbon sources while stepwise increasing thetemperature, pressure, salinity and pH. The resulting modified ormetabolically weaned thermophilic bacteria exhibit different chemicaland biochemical properties than the thermophilic bacterial strainsinitially isolated from unique geothermal locations in the South Pacificand North America. Microorganisms which survive essentially on oil underextreme conditions of temperature, pressure, salinity and pH includeAchromobacter sp. BNL-4-23 (ATCC 55021), Sulfalobus solfataricusBNL-TH-29 (ATCC 55022), Sulfalobus solfataricus BNL-TH-31 (ATCC 55023),Sulfolobus acidocaldarius BNL-TH-1 (ATCC 35091), Pseudomonas sp.BNL-4-24 (ATCC 55024), Leptospirillum ferrooxidans BNL-5-30 (ATCC53992), Leptospirillum ferrooxidans BNL-5-31 (ATCC 53993), Acinetobactercalcoaceticus BNL4-21 (ATCC 53996), Arthrobacter sp. BNL4-22 (ATCC53997).

The microorganisms which can feed on essentially crude oil are thenisolated and are further nutritionally stressed to metabolize complexmacromolecular compounds found in solid carbonaceous material such ascoal. The nutritional stressing on a coal substrate is also conductedstepwise under challenged growth conditions including a temperaturerange from about 40° C. to about 85° C. reaction range pressure rangefrom about ambient pressure to about 2500 p.s.i., a pH range from about2 to about 10, a salinity range from about 1.5 weight % to about 35weight % and a toxic metal concentration from about 0.01 weight % toabout 10 weight %.

Microorganisms which can survive essentially on solid carbonaceousmaterial such as coal under challenge growth conditions includeAcinetobacter calcoaceticus, BNL-4-21s (ATCC 55489), Arthrobacter sp.BNL4-22s (ATCC 55490), Achromobacter sp. BNL-4-23s (ATCC 55491),Pseudomonas sp. BNL-4-24s (ATCC 55492), Mixed Culture R.I. -1 (ATCC55501), Leptospirillum ferrooxidans BNL-5-30s (ATCC 55523),Leptospirillum ferroxidans BNL-5-31s (ATCC 55524), Thiobacillusferroxidans BNL-2-44s (ATCC 55525), Thiobacillus ferrooxidans BNL-245s(ATCC 55526), Thiobacillus ferrooxidans BNL-2-46s (ATCC 5 55527),Thiobacillus ferrooxidans BNL2-47s (ATCC 55528), Thiobacillusferrooxidans BNL-2-48s (ATCC 55529), Thiobacillus ferrooxidans BNL-2-49s(ATCC 55530), Unknown BNL-NZ-3 (ATCC 55488), Unknown BNL-NZ-5 (ATCC tobe determined), Mixed Culture R.I.-2 (ATCC 55502), Mixed Culture R.I.-3(ATCC 55503), Mixed Culture R.I.-4 (ATCC 55504), Mixed Culture R.I.-5(ATCC 55505), Mixed Culture R.I.-6 (ATCC 55506), Mixed Culture R.I.-7(ATCC 55507), Mixed Culture R.I.-8 (ATCC 55508), Mixed Culture R.I.-9(ATCC 55509), Mixed Culture R.I.-10 (ATCC 55510), Mixed Culture R.I.-11(ATCC 55511), Mixed Culture R.I.-12 (ATCC 55512), Mixed Culture R.I.-13(ATCC 55513), Mixed Culture R.I.-14 (ATCC 5554) and mixtures thereof.

The modified microorganisms metabolically weaned by nutritionalstressing under challenged growth conditions are also encompassed in thepresent invention.

As a result of the present invention a process for the biochemicaltransformation of solid carbonaceous material is provided which can beused very effectively to break down the complex hydrocarbon structure ofcoal to provide a biochemically transformed coal which is cleaner andricher in low molecular weight hydrocarbon fragments. The biotransformedcoal is a more suitable feedstock for liquefaction, gasification and/orcombustion processes. The metabolically weaned microorganisms of thepresent invention are capable of cleaving selectively macromolecularcompounds found in solid carbonaceous materials at hetero atom sitesthereby providing a coal which has been depolymerized, desulfurized anddemineralized. As a result, the biochemically transformed coal obtainedby applying the process of the present invention also lowers operationcosts of coal liquefaction, gasification, combustion and other coalutilizations.

Other improvements which the present invention provides over the priorart will be identified as a result of the following description whichsets forth the preferred embodiments of the present invention. Thedescription is not in any way intended to limit the scope of the presentinvention, but rather only to provide a working example of the presentpreferred embodiments. The scope of the present invention will bepointed out in the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a typical chemical structure of bituminous coal.

FIG. 2 is a pyrolysis-gas chromatography-photometric detector analysisof sulfur content for North Dakota lignite and Kentucky No. 8 coaluntreated and treated with modified strains of BNL-TH-29, BNL-NZ-3 andBNL-NZ-5 bacteria.

FIG. 3 is a pyrolysis-gas chromatography-mass spectroscopy analysis ofalkanes for Kentucky No. 8 bituminous coal and North Dakota lignite,untreated and treated with modified strains BNL-NZ-5 bacteria.

FIG. 4 shows x-ray absorption near edge structure spectroscopy (XANES)of Kentucky No. 8 bituminous coal, untreated and treated with modifiedstrains of BNL-TH-29, BNL-NZ-3 and BNL-NZ-5 bacteria.

DEPOSIT

A number of biologically pure microorganisms, modified following theprocedures of the present invention and illustrative of the modifiedthermophilic microorganisms useful in the process of the presentinvention have been deposited in the American Type Culture Collectionprior to the 10801 University Boulevard, Manassas, Va. 26110-2209 filingdate of this application in accordance with the permanency andaccessibility requirements of the U.S. Patent and Trademark Office. Thefollowing is a list of the deposited microorganisms:

Applicants' ATCC Scientific Description Reference DesignationAchromobacter sp. BNL-4-23 55021 Sulfolobus solfataricus BNL-TH-29 55022Sulfolobus solfataricus BNL-TH-31 55023 Pseudomonas sp. BNL-4-24 55024Leptospirillum ferrooxidans BNL-5-30 53992 Leptospirillum ferrooxidansBNL-5-31 53993 Acinetobacter calcoaceticus BNL-4-21 53996 Arthrobactersp. BNL-4-22 53997 Acinetobacter calcoaceticus BNL-4-21s 55489Arthrobacter sp. BNL-4-22s 55490 Achromobacter sp. BNL-4-23s 55491Pseudomonas sp. BNL-4-24s 55492 Leptospirillum ferrooxidans BNL-5-30s55523 Leptospirillum ferrooxidans BNL-5-31s 55524 Thiobacillusferrooxidans BNL-2-44s 55525 Thiobacillus ferrooxidans BNL-2-45s 55526Thiobacillus ferrooxidans BNL-2-46s 55527 Thiobacillus ferrooxidansBNL-2-47s 55528 Thiobacillus ferrooxidans BNL-2-48s 55529 Thiobacillusferrooxidans BNL-2-49s 55530 Unknown BNL-NZ-3 55488 Unknown BNL-NZ-5 tobe determined Mixed Culture R.I.-1 55501 Mixed Culture R.I.-2 55502Mixed Culture R.I.-3 55503 Mixed Culture R.I.-4 55504 Mixed CultureR.I.-5 55505 Mixed Culture R.I.-6 55506 Mixed Culture R.I.-7 55507 MixedCulture R.I.-8 55508 Mixed Culture R.I.-9 55509 Mixed Culture R.I.-1055510 Mixed Culture R.I.-11 55511 Mixed Culture R.I.-12 55512 MixedCulture R.I.-13 55513 Mixed Culture R.I.-14 55514

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for the biochemicaltransformation of solid carbonaceous material. More specifically, themethod includes treating a slurry of coal with modified biological purestrains of thermophilic microorganisms which are capable ofdepolymerizing, desulfurizing and/or demineralizing coal by selectivelycleaving molecular structures at organic carbon-carbon sites,carbon-sulfur sites and other heteroatom sites including metals andnitrogen.

In the complex polymeric structure of coals, organic sulfur is believedto be bound primarily in crosslinked thiophenic rings, thiols, sulfides,disulfides bridges, and sulfones. The complex coal structure frequentlyencloses pyrites, sulfates, and elemental sulfur. A typical chemicalstructure of bituminous coal is shown in FIG. 1.

“Metabolically weaned” as used in the present invention refers to themicroorganism or microorganisms which have been adapted to metabolizesolid complex macromolecular compounds found in solid carbonaceousmaterials. This metabolization includes, for example, depolymerization,desulfurization, and demineralization of solid carbonaceous materialssuch as coal.

As used in the present invention depolymerization refers to thedecomposition of complex macromolecular compounds found in solidcarbonaceous materials to lower molecular weight hydrocarbons residues.Desulfurization refers to the removal of sulfur from complexmacromolecular compounds found in solid carbonaceous material.Desulfurization also results in the formation of hydrocarbons of lowermolecular weight and other simpler organic compounds. Demineralizationis used to refer to the removal of trace metals by microorganismscapable of metabolizing organometallic compounds found in solidcarbonaceous materials. Thus, the result of biochemical treatment ofsolid carbonaceous material, such as coal, with the challengedmicroorganisms of the present invention is a coal matrix which hassimpler hydrocarbons and is lower in sulfur and toxic metal content. Theresulting transformed matrix undergoes combustion, liquefaction, orgasification much more efficiently and with the formation of fewerenvironmentally undesirable byproducts.

The biochemical transformation process of the present invention isbroadly applicable to the treatment of various types ofsulfur-containing solid carbonaceous material. One type ofsulfur-containing solid carbonaceous material is coal. Coals which canbe treated by the process of the present invention include anthracite,bituminous, subbituminous, mine tailings, fines, lignite and the like.Other finely divided solid carbonaceous solids, such as coke may alsobenefit from the present process. Preferably, the coal subjected to thepresent biochemical transformation process is present as a slurry in aparticulate form. Thus, in carrying out a preferred embodiment of thepresent invention, typically raw mined coal is first reduced to asmaller particle size preferably less than but not limited to about 100mesh. The coal particulates are then formed into a slurry with watersuch that the solids concentration in the slurry is from about 1% toabout 60% by weight.

In a preferred embodiment, the slurry of the carbonaceous materialincludes bituminous Kentucky No. 8 coal and/or North Dakota lignite. Theelemental analysis for North Dakota lignite and Kentucky No. 8bituminous coal is set forth in Table 1 below.

TABLE 1 Elemental Analysis of Coal North Dakota Kentucky No. 8 lignitebituminous Carbon 61.08 78.3 Hydrogen 4.08 3.3 Oxygen* 23.44 4.6Nitrogen 0.87 1.8 Sulfur 0.53 1.1 Ash 10.00 10.095 *By difference

In accordance with the present invention, modified microorganisms areproduced for use in biochemical transformation of solid carbonaceousmaterial. Selected thermophilic microbial strains isolated from uniquegeothermal locations in the South Pacific and North America aremetabolically weaned and forced to adapt to growth on an oil substrateat a desired temperature, pressure, pH and salinity. The thermophilicbacteria which survive growth on an essentially oil substrate are thenadapted to grow on a solid carbonaceous material substrate, such as coalat a desired temperature, pressure, pH, salinity and toxic metalconcentration.

An aqueous coal slurry is inoculated with modified strains of selectedmicroorganisms. A nutrient medium is generally added to the aqueousslurry in order to provide the source of metabolites. A typical nutrientmedium comprises sodium chloride, potassium phosphate, magnesium sulfateand a source of casein. The temperature at which the biochemicaltransformation process is carried out is generally in a range from about40° C. to about 85° C. The pH is maintained in a range from about 2 toabout 10. A final pH of about 4 is preferred. The pressure range ismaintained from about ambient to about 2500 p.s.i. The modified strainsused in the process of the present invention are adapted to toxic metalconcentrations from about 0.01 weight % to about 10 weight %, dependingon the metal. The strains useful for the biotreatment of carbonaceousmaterial are also adapted to elevated concentrations of salinity fromabout 1.5 weight % to about 35 weight %.

The original microorganisms employed in the present invention have beenisolated from geothermal sources and have been “metabolically weaned” tometabolize solid carbonaceous material. Just as offspring must becomeaccustomed to food other than maternal nourishment, thermophilicmicroorganisms are grown in a challenged environment so that they areforced to adapt to metabolizing solid carbonaceous material. After themicroorganisms have been isolated, they are initially grown in a mediumcontaining crude oil supplemented with other sources of nutrients, suchas minerals and more easily metabolized sources of energy such asmolasses and yeast extract. The strains which can survive in thepresence of crude oil are then nutritionally stressed by subjecting themto further challenge by selection under more extreme conditions.

“Nutritionally stressing” as used in the present invention means toforcedly adapt the microorganism to metabolize a source of nutritionwhich it does not normally metabolize during growth. This has beenaccomplished with respect to the present invention by a “challengedgrowth” process. The selection processes proceed by removal of the moreeasily metabolizable carbon sources, i.e., the molasses, yeast extract,oil and the like while increasing stepwise the concentration of coal andanyone of the other conditions such as temperature, pressure, pH,salinity and toxic metals content.

In a challenged growth process the thermophilic microbial strain orstrains are grown in a medium containing crude oil supplemented withcarbon sources other than carbonaceous materials and nutrients otherthan crude oil at a selected temperature, pressure and salinityconcentration. The surviving thermophilic microbial strains are isolatedand grown in a medium containing increased amounts of crude oil anddecreasing amounts of the carbon source which is other than thecarbonaceous materials at a temperature, pressure and salinity higherthan in the previous step. The previous step is then repeated and ineach successive step the medium contains increased amounts of crude oiland decreasing amounts of the carbon source which is other thancarbonaceous materials and at a temperature, pressure and salinityhigher than in each previous step until microbial strains are obtainedthat are capable of growing essentially on crude oil as a carbon sourceand at a temperature range from about 40° C. to about 85° C. at apressure range from about ambient to about 2500 p.s.i. and the salinityrange from about 1.3 weight % to about 35 weight %. The thermophilicmicrobial strains which are able to survive growth on essentially crudeoil are then selected and grown in a medium containing decreasingamounts of oil and increasing amounts of coal as a carbon source at afirst chosen condition selected from the group consisting oftemperature, pressure, pH, salinity and toxic metal concentration,wherein the chosen condition is higher than in the previous step. Thenutritional stressing on coal is repeated until the medium contains ahigher amount of coal as a carbon source at a successive chosencondition higher than in the previous step until the microbial strain ormixture of microbial strains is developed which is capable of growing anessentially coal as a carbon source and at a final chosen conditionselected from the group consisting of temperature, pressure, pH,salinity and a toxic metal concentration. The challenged growthtemperature for nutritional stressing on a coal substrate is from about40° C. to about 85° C., the pressure is from about ambient to about 2500p.s.i., pH is from about 2 to about 10, the salinity is from about 1.5weight % to about 35 weight % and the toxic metal concentration is fromabout 0.01% weight % to about 10% weight %. Methods of isolation andculturing have been described in detail in U.S. application Ser. No.571,917 filed Aug. 24, 1990 and U.S. application Ser. No. 905,391 filedJun. 29, 1992, the contents of which are incorporated herein byreference as if set forth in full. Thus, the microorganisms of thepresent invention are metabolically weaned to metabolize solidcarbonaceous material under challenged growth conditions.

In addition to thermophilic bacteria strains which have been engineeredto survive a harsh variety of environmental conditions present in oilreservoirs and which have been useful in microbially enhanced oilrecovery (“MEOR”), other strains of thermophilic bacteria have beendeveloped which have been useful for the biochemical transformation ofsolid carbonaceous materials, such as coal. As used in the presentinvention, thermophilic bacteria refer to bacteria which thrive attemperatures exceeding 40° C.

Table 2, which follows, lists biologically pure strains of thermophilicbacteria produced through challenged growth processes, which modifiedmicroorganisms are useful for biochemical transformation of carbonaceousmaterial including oil and coal. In Column “a” of table 2 there arelisted the cultures which were subjected to challenged growth processesby the source of the original culture. In column “b” the American TypeCulture Collection No. or “ATCC No.” is set forth. In column “c” theBrookhaven National Laboratory No. or “BNL No.” is listed.

TABLE 2 Microorganisms Produced Through Challenged Growth (a) (c)Original Culture (b) Modified Designation Source/DepositoryMicroorganism Scientific Description ATCC NO. BNL No. 1. Achromobactersp. 55021 BNL-4-23 2. Sulfolobus solfataricus 55022 BNL-TH-29 3.Sulfolobus solfataricus 55023 BNL-TH-31 4. Pseudomonas sp. 55024BNL-4-24 5. Leptospirillum ferrooxidans 53992 BNL-5-30 6. Leptospirillumferrooxidans 53993 BNL-5-31 7. Acinetobacter calcoaceticus 53996BNL-4-21 8. Arthrobacter sp. 53997 BNL-4-22 9. Acinetobactercalcoaceticus 55489 BNL-4-21s 10. Arthrobacter sp. 55490 BNL-4-22s 11.Achromobacter sp. 55491 BNL-4-23s 12. Pseudomonas sp. 55492 BNL-4-24s13. Leptospirillum ferrooxidans 55523 BNL-5-30s 14. Leptospirillumferroxidans 55524 BNL-5-31s 15. Thiobacillus ferroxidans 55525 BNL-2-44s16. Thiobacillus ferrooxidans 55526 BNL-2-45s 17. Thiobacillusferrooxidans 55527 BNL-2-46s 18. Thiobacillus ferrooxidans 55528BNL-2-47s 19. Thiobacillus ferrooxidans 55529 BNL-2-48s 20. Thiobacillusferrooxidans 55530 BNL-2-49s 21. Unknown 55488 BNL-NZ-3 22. Unknown tobe determined BNL-NZ-5 23. Mixed Culture 55501 R.I.-1 24. Mixed Culture55502 R.I.-2 25. Mixed Culture 55503 R.I.-3 26. Mixed Culture 55504R.I.-4 27. Mixed Culture 55505 R.I.-5 28. Mixed Culture 55506 R.I.-6 29.Mixed Culture 55507 R.I.-7 30. Mixed Culture 55508 R.I.-8 31. MixedCulture 55509 R.I.-9 32. Mixed Culture 55510 R.I.-10 33. Mixed Culture55511 R.I.-11 34. Mixed Culture 55512 R.I.-12 35. Mixed Culture 55513R.I.-13 36. Mixed Culture 55514 R.I.-14

From the modified microorganisms listed in Table 2 the first eight,namely BNL-4-23, BNL-TH-29, BNL-TH-31, BNL4-24, BNL-5-30, BNL-5-31,BNL4-21, BNL4- 22 have been challenged or adapted on oil substrates andincreasing temperatures, pressures, pH and salinity as set forth in U.S.application bearing Ser. No. 08/160,417, the contents of which areincorporated herein by references as if set forth in full. The remainingmicroorganisms listed in Table 2, have been initially challenged oncrude oil substrates, and then have been adapted in successive steps inmedia containing decreasing amounts of crude oil and increasing amountsof coal at one selected condition chosen from temperature, pressure,salinity, pH and toxic metal content wherein each successive selectedcondition is higher than in the previous step. For example, the salinityis gradually increased from about 1.5 weight % to about 35 weight %,wherein 15% weight is preferred.

The biochemical treatment of carbonaceous material can be caused by asingle biologically pure strain of modified microorganism or mixedcultures of mutated biologically pure microorganisms which are usedsubsequently under either aerobic or anaerobic conditions depending onthe ranges of salinity, toxic metals concentrations, pH, temperaturesand pressures present when the solid carbonaceous material is treated.It is possible to maximize the effect of the biochemical transformationof solid carbonaceous material by using a combination of organisms,wherein each of which is very efficient in producing one or more of thedesired degradations. For example, a mixed culture could includemodified organisms which cleave the coal matrix very efficiently atheterosites, thereby causing the depolymerization to lighterhydrocarbons. The same mixture could include modified organisms whichare efficient at desulfurizing and/or demineralizing of the coal matrix.The mixed culture approach permits tailoring of the microbial packageused for biochemical transformation of different types of solidcarbonaceous material.

For the purposes of the present invention, the preferred microorganismsare the thermophilic archaebacteria of the Sulfolobus species which havebeen modified by challenged growth processes. Most preferred areBNL-TH-29, BNL-NZ-3 and BNL-NZ-5 which were obtained from the parentstrains of Sulfolobus Solfataricus (ATCC 35091), Unknown (ATCC 55488)and Unknown (ATCC to be determined). These modified bacterial strainsare especially suitable for the processes of the present inventionbecause they remain viable over extended periods, often up to six (6)months under harsh environmental conditions including temperatures in arange of 40° C. to about 85° C., high pressures in a range from aboutambient to about 2500 p.s.i., pH range from about 2 to about 10, toxicmetal concentration in a range from about 0.01 wt % to about 10 wt %,and salinity in a range from about 1.5 wt % to about 35 wt %.

Analysis of the solid carbonaceous material treated with the modifiedmicroorganisms of the present invention at a temperature range of fromabout 40° C. to about 85° C. and a pressure range of ambient-2500 p.s.i.indicates that as a result of biotreatment, the carbonaceous materialbecomes depolymerized and desulfurized and shows significantly decreasedtrace metal content.

In the examples which follow, bioreactors were used for metabolicallyweaning the microorganisms by challenged growth procedures in a 40°-85°C. water bath at total pressures ranging from ambient to 2500 p.s.i.,under carbon dioxide and nitrogen in a volume ratio of 1:25, at 70° C.Minibioreactors are used for anaerobic pressurized experiments whileconventional culture flasks are used for aerobic experiments at elevatedtemperatures.

The medium for the challenged growth procedures includes inorganicsalts, e.g., (NH₄)₂SO₄, MgSO₄, KH₂PO₄, crude oil, yeast extract and coalas a source of carbon. Incubations of cultures can be carried out underdifferent pressures, gas compositions and temperatures. Yeasts, molassesand sources of carbons other than crude oil are used in conjunction withcrude oil only at initial stages of growth. The organism is allowed togrow to a steady concentration, i.e. 1×10⁸/ml under conditions in whichthe concentration of oil is increased and the other sources of carbonare decreased. During this initial stage, the organism is maintained atelevated temperatures and pressures. Generally, if the organism growssuccessfully to the desired level in the presence of crude oil as thesole source of carbon, but only at ambient temperatures, then it is“challenged” stepwise to higher temperatures and pressures until steadygrowth and desired concentrations are achieved. Two or three transfersat optimum conditions of growth often suffice to generate a modifiedmicroorganism suitable for microbially enhanced oil recovery (“MEOR”).

The modified microorganisms challenged as described above are thenisolated and further adapted to growth on coal, increased salinity andtoxic metal content. This process is accomplished by incubating themicroorganisms suitable for MEOR in media of decreasing oil andincreasing coal concentration as a source of carbon and such toxicmetals as ordinarily found in coal. This process is also accomplished instepwise manner, i.e., when the organism grows successfully in thepresence of coal as the sole source of carbon, but only at ambienttemperatures, then it is further “challenged” stepwise to highertemperatures, pressures, salinity and toxic metal contraction untilsteady growth and desired concentrations are achieved. Thus, thetemperature is increased from about 40° C. to about 85° C. The pressureis increased from about ambient to about 2500 p.s.i. The pH is increasedfrom about 2 to about 10. The salinity is also increased from about 1.5wt % to about 35 wt % sodium chloride, wherein a concentration of 15% byweight is preferred. The toxic metal content is increased from about0.01 wt % to about 10 wt %, depending on the metal.

The determination of organic sulfur content of coal is conventionallymeasured as the difference between total sulfur and inorganic sulfur.The results are often subject to large errors inherent to the methodused for the determination of elemental sulfur, sulfate and pyrite incoal. More recently, pyrolysis-gas chromatography-mass spectroscopy(“PY-GC-MS”) and x-ray absorption near edge structure spectroscopy(“XANES”) have been used as sensitive analytical tools for thedetermination of organically bound sulfur in kerogen and asphalteneswhich chemically resemble coal. In the present invention, untreated andbiotreated coal samples have been analyzed first by PY-GC-MS and then byXANES methods.

In the examples which follow, the efficacy of the present invention isclearly shown. Two independent analytical methods were used to analyzecoal which has undergone biochemical transformation in accordance withthe process of the present invention. These analytical methods arepyrolysis-gas chromatography-mass spectrometry (“PY-GC-MS”) and x-rayabsorption near edge structure spectroscopy (“XANES”). For example, asshown in FIG. 2 and Table 4 herein an analysis of biochemicallytransformed lignite and bituminous coals treated with modified,biologically pure thermophilic bacteria BNL-TH-29, BNL-NZ-3 and BNL-NZ-5shows a reduction of the sulfide content and the content of C1 to C5substituted thiophenes. In addition, as shown in FIG. 3 hereto PY-GC-MSanalysis of biotreated and untreated bituminous coal indicates thatduring the biochemical treatment of coal a biochemical transformationtakes place during which the molecular structure of coal is cleaved athetero atom sites such as sulfur, nitrogen and metals so that thetreated coal contains more small to medium molecular weight fractions inpreference to high molecular weight residues in terms of scans number(low number, e.g. 600, corresponds to molecular weights of about C6 toC10 hydrocarbons and progressively increasing reaching a molecularweight range of above C30 hydrocarbons in the 3000 scans number).Without wishing to be bound by any theory it appears that duringbiotreatment of solid carbonaceous material, the overall breakage ofsulfide linkages leads to an extensive breakdown of crosslinking in thestructure of the carbonaceous material.

During the biochemical transformation of coal the removal of organicsulfur is often accompanied by the removal of amounts of trace metals.The trace metals found in coal are often toxic. Metals removed bybiochemical treatment of coals include vanadium, manganese, copper,strontium, yttrium, zirconium, lanthanum, cerium, lead, thorium anduranium.

EXAMPLES

The following examples have been carried out to show the effect ofdifferent strains of modified microorganisms on solid carbonaceousmaterial. These examples also serve to provide further appreciation ofthe invention but are not meant in any way to restrict the effectivescope of the invention.

Example 1 Isolation and Culturing Methods of Thermophilic Bacteria

Thermophilic bacteria NZ and TH capable of hydrocarbon degradation wereisolated from unique geothermal locations located in the South Pacificand North America, respectively. Because both cultures were isolatedfrom geothermal sources, they grew well in temperatures ranging from 65°C. to 80° C. For example, a 5% (vol./vol.) inoculum reached stationaryphase growth in two days at concentrations about 1×10⁸ ml⁻¹.

The medium formula used for NZ was 1 g pancreatic digest casein, 0.05 gsodium thioglycollate, 2.5 g NaCl, 0.25 g CaCl₂, 1.57 g K₂HPO₄, 0.891 gKH₂PO₄, 0.4 g (NH₄)₂ SO₄ and 0.25 g MgSO₄ in a liter of water. Theformula for TH medium was 1 g yeast extract, 1 g casamino acids, 3.1 gKH₂PO₄, 2.5 g (NH₄)₂SO₄, 0.2 g MgSO₄7H₂O, 0.25 g CaCl₂2H₂O, 0.22 mgZnSO₄7H₂O, 0.05 mg CuCl₂2H₂O, 0.03 mg Na₂MoO₄2H₂O, 0.03 mg VOSO₄2H₂O and0.01 mg CaSO₄7H₂O in a liter of distilled water. The final pH wasadjusted to 4.0 by adding H₂SO₄. Three strains of gram positive rodsfrom these isolates were used in this study. A detailed description ofisolation and culturing methods for thermophilic NZ and TH bacteria isset forth in commonly-owned U.S. application bearing Ser. No. 07/905,391filed Jun. 29, 1992, the contents of which are incorporate herein as ifset forth in full.

Example 2 Biotreatment of Coals

Powdered coals were stored in a desiccator without any special effort tosterilize samples prior to executing the experiments. Two per cent (w/v)of the powdered coal (100 mesh) was cultured with selected bacteria fora week in an Erlenmeyer flask without shaking at 65° C. Control sampleswere subject to identical culture conditions without, however, addedbacterial inoculum. After treatment, the reaction mixtures were filteredthrough Whatman No. 1 filter paper and washed twice, each timesubmersing the filtered coal in 100 ml of distilled water per 2 g sampleand shaken vigorously. The reaction mixtures were then filtered throughWhatman No. 1 filter paper again. Finally, the samples were air driedand stored in a desiccator at room temperature. The samples were driedto constant weight for further analysis. About 2.5-3.0 mg of dried coalsamples were analyzed by a Perkin-Elmer Model 240C elemental analyzer inaccordance with procedures published in a report by Perkin-ElmerInstrument Co. entitled “CHN Analysis of Coal with the Perkin-ElmerModel 240 Elemental Analyzer and Sulfur Analysis Kit for the Model 240Elemental Analyzer, Report No. 993-941.7, Danbury, Conn., 1980. Theanalyses involving coal with 1%, or less, sulfur content were calibratedagainst standards and untreated coal as illustrated in Table 3 below.The results were reported as averages of triplicate measurements.

TABLE 3 TOTAL SULFUR REMOVAL (%) NORTH KENTUCKY DAKOTA NO. 8 LIGNITEBITUMINOUS TREATMENT COAL COAL 1. Control (a) (Inorganic sulfur removal)11 ± 2 10 ± 1 2. BNL-NZ-3 (Both Inorganic and Organic Sulfur Removal) 18± 3 12 ± 1 3. Organic Sulfur Removal by BNL-NZ-3 = 2-1 7 ± 2 2 ± 1 4.BNL-NZ-5(Both Inorganic and Organic Sulfur Removal) 24 ± 3 25 ± 2 5.Organic Sulfur Removal by 13 ± 2 15 ± 2 BNL-NZ-5 = 4-1 6. Control (b)(Inorganic Sulfur Removal) 19 ± 3 20 ± 2 7. BNL-TH-29 (Both Inorganicand Organic Sulfur Removal) 29 ± 4 30 ± 2 Organic Sulfur RemovalBNL-TH-29 = 7-6 10 ± 3 10 ± 2 (a) (b) The difference in sulfur removalvalues for controls is due to different media used for the growth oforganisms. The media for control (b) was more acidic than for control(a) and therefore more inorganic sulfur was also removed.

The results set forth in Table 3 show that the biotreatment ofbituminous coal No. 8 from Kentucky and lignite coal from North Dakotawith biologically pure thermophilic strains of BNL-TH-29, BNL-NZ-3, andBNL-NZ-5 resulted in a reduction of the total sulfur content. Incomparison, there was a small reduction of sulfur content observed inthe control experiments, which is attributed to the action of indigenousbacteria present in coal and the dissolution of inorganic sulfur formssuch as sulfates at 65° C. The additional total sulfur reduction wassubstantial indicating that the process of the present invention is veryeffective for organic sulfur removal and as a result for coaldesulfurization.

In another embodiment, prior to biotreatment, the coal sample is washedwith dilute nitric acid to remove the inorganic sulfur. For example,dilute nitric acid treatment was used to remove pyrite and other formsof inorganic sulfur in coal. Coal samples of 10 g of 150-200 mesh weresuspended in 100 ml of 2N nitric acid and kept at room temperature for14 hours. The acid treated coals were then thoroughly washed with 200 mlof distilled water. They were then filtered, air dried, and kept in adesiccator at room temperature for further biotreatment.

Example 3 PY-CG-MS Analysis

About 2.00 mg desiccator dried coals as obtained in Example 2 werepyrolyzed in a stream of pure helium having less than 1 ppm air in aChemical Data System model 190 pyroprobe. The pyroprobe was programmedat a heating rate of 10,000° C./sec to 660° C. for 20 seconds, and thepyrolysate was swept directly into a Perkin Elmer 8700 Gas Chromatograph(“GC”) with a J & W DB-1 capillary column 30 mm in length, 0.25 mm I Dand 1 μm film. The GC temperature program was 40° C. for 5 minutes whichwas then increased to 320° C. for 25 minutes at a heating rate of 8°C./minute. The GC system was equipped with a splitter which allowed theeffluent leaving the column to be simultaneously analyzed by a FlamePhotometric Detector (FPD) and by a mass selective Ion Trap Detector(ITD). The FPD was set on fast response and non-linearization mode,whereas the ITD was set on mass scan of 40 to 500 Daltons at a rate ofone scan per second. The results are presented in FIGS. 2 and 3 hereto.The composition of the chromatogram were identified by the use ofstandards and by means of EPA/NIH mass spectrometric data base librarysearch. Some of the peaks identified in FIG. 2 are listed in Table 4.

TABLE 4 Peaks listed in FIG. 2 1 H₂S and other sulfur gases 2 Thiophene3 C1 substituted thiophenes 4 C4-C8 sulfides 5 C3-C6 mercaptans 6Unidentified sulfur compounds 7 Unidentified sulfur compounds 8 C2substituted thiophenes 9 C3 substituted thiophenes 10 C4 substitutedthiophenes 11 C5 substituted thiophenes 12 Benzothiophenes 13 C1substituted benzothiophenes 14 C2 substituted benzothiophenes 15Bithiophenes 16 Dibenzothiophenes

The peaks shown in FIG. 2 and identified in Table 4 indicate that thetreatment of lignite coal with metabolically weaned thermophilicbacteria BNL-TH29, BNL-NZ-3 and BNL-NZ-5 reduced sulfides and C1 to C5substituted thiophenes. The results shown in FIG. 2 indicate substantialdegradation of sulfur linkages and thiophenic rings caused by thebiochemical treatment process of the present invention. The biotreatmentresults shown in FIG. 2 for bituminous coal are somewhat different fromthose obtained for lignite coals. PY-GC-FPD analyses illustrated in FIG.2 show that there was a substantial degradation of the thiophenic ringtype compounds with minor changes in the sulfide forms.

The graphs set forth in FIG. 2 show some noise or spurious signals knownas artifacts. Pyrolysis of macromolecular substances such as coals andheavy crude oils are known to be subject to artifact formation. Recentstudies of pyrolysis of fossil polymers at low temperature, i.e., 610°C. have indicated that products are formed mainly by cleavage of onebond at a time. Possible mechanisms of biochemical degradation oforganic sulfur compounds by thermophiles may also involve a stepwiseenzymatic breakdown of organic sulfur structures as found in kerogens.One example of such biochemical degradation is the flash pyrolysisproduct of alkylthiophenes as derivatives of their correspondingalkylthiophene moieties in kerogen. The results shown in FIG. 2 supportthe possible mechanisms of biodegradation discussed above.

The results set forth in FIG. 3 are discussed by reference tobiodegradation of model organosulfur compounds (“OSC”). Thebiodegradation of OSC has been extensively reviewed by Fedorak, F. M. in“Geochemistry of Sulfur in Fossil Fuels, ACS Symposium Series 429,American Chemical Society, Washington, D.C., 93-112, 1990. In general,the biochemical reactions of model OSC leads to the biodegradation ofthiophenic compounds yielding hydroxy substituted rings, as well as openring sulfones or sulfoxides. If similar biochemical reactions occurduring the biotreatment of coals, the reduced sulfur forms may lead to abiochemical partial breakdown of coal structure observable in coalpyrolysis. Analysis of biotreated and untreated bituminous and lignitecoal by PY-GC-MS as illustrated in FIG. 3, shows that the biotreatedcoal contained more small to medium molecular weight fractions versushigh molecular weight residues. The effect is more pronounced in thebiotreated lignite. It appears that the overall breakage of sulfidelinkages leads to an extensive breakdown of crosslinking in lignite coalstructure. As a result, such biochemical reactions may be useful in thepre-treatment of coal prior to mild gasification of coal as well asbiodesulfurization of coal.

Example 4 X-Ray Absorption Fine Structure Spectroscopy Analysis

X-ray absorption spectroscopy measurements were performed at theNational Synchrotron Light Source (“NSLS”) X-19A beam line at BNL. Thex-ray beam emitted from the storage ring was collimated by an adjustablevertical slit and was diffracted by a double crystal monochromator[Si(111)], which passed a narrow energy band. The overall energyresolution was estimated to be 0.5 eV at 2500 eV photon energy. Harmonicrejection was accomplished by slightly misaligning the crystalorientation so that the intensity of the monochromator Bragg peak wasreduced by about 80%. A Stern-Heald type fluorescence detector asdescribed in Scientific Instrumentation, 50, 1579 (1979) placed at 90°to the beam was used to measure the fluorescent x-ray which was emittedin the relaxation of the core hold. For accurate measurements of edgeshift, the spectrometer was calibrated so that the white line maximumpeak of the elemental sulfur correspond to 2472.1 eV. Spectra wererecorded in four energy regions about the edges: 2.32 keV to 2.46 keV in2 eV steps; 2461 eV to 2500 eV in 0.08 eV steps; 2501 eV to 2520 eV in0.2 eV steps; and 2521 eV to 2800 eV in 3 eV steps. The samples ofpowdered coal (100 mesh) were held in the cell of a 6 μm thickpolypropylene bag.

Due to the self-absorption effect the attenuated fluorescence spectrarequired correction. The method developed by Waldo and Penner-Hahn asdescribed in Waldo, G. S. et al., Geochimica et Cosmochimica Acts, 55,801-814 (1991) was adopted. Following this procedure, the data wasnormalized in the post-edge region to fit a tabulated x-ray absorptioncross section known as McMaster table as more particularly described inMcMaster, W. H. et al. UCRL-50174, Sec II, Rev. 1, Natl. Techn. Inf.Serv., Springfield, Va. (1969). The normalized spectra were fitted withlinear combinations of absorption spectra of a model compounds ofsulfur, sulfide, disulfide, thiophene, sulfone, and sulfate using anon-linear least-squared procedure. The fitting range (2465-2485 eV) wasselected so that the region contained the white-line maxima for all ofthe model compounds. The adjustable parameters for each model spectrumwere a scaling factor and an optional energy offset. Typically, energyoffset was less than 0.2 eV and not greater than 0.4 eV. The scalingfactor directly reflected the quantitive amount of each sulfur form.XANES analyses of Kentucky bituminous samples are presented in FIG. 4.These analyses were used together with the results from Table 3 tocalculate the percentage of each sulfur form as presented in Table 5below. Each group of sulfur formed in the coal was calculated as molepercentage of total sulfur forms.

TABLE 5 XANES Analysis of Biotreated Kentucky No. 8 Bituminous Coal^(d)Sulfides Thiophenes Sulfoxides Sulfones Sulfates BNL-NZ Control^(b)0.240 0.441 0.121 0.045 0.153 BNL-NZ-3 Treated 0.234 0.408 0.086 0.0470.194 BNL-NZ-5 Treated 0.208 0.296 0.089 0.053 0.179 BNL-TH-29^(a)Control^(c) 0.184 0.303 0.109 0.055 0.239 BNL-TH-29 0.198 0.198 0.1770.045 0.173 ^(a)Data have been normalized to 1.1% total sulfur contentfor untreated samples ^(b)Control for NZ Medium ^(c)Control for THMedium ^(d)Mole percentage of total sulfur form

The XANES analysis of Kentucky No. 8 coal resolved the sulfurdistribution in terms of a combination of representative groups ofsulfur standards as illustrated in FIG. 4 and Table 5. A comparison ofdata for treated and untreated coal samples showed a significantdecrease in thiophenic sulfur forms for bituminous coal treated withBNL-NZ-3, BNL-NZ-5 and BNL-TH-29 in a range from about 3.3 mol % toabout 14.5 mol %.

The treatment of bituminous coal with both BNL-NZ-3 and BNL-NZ-5resulted in a significant reduction of thiophenic sulfur and a smallerreduction of sulfoxide sulfur forms with small increases of sulfone andsulfate forms of sulfur. The increase in sulfones and sulfates is theresult of bacterial oxidation of some of the sulfur compounds found incoal to sulfur oxide compounds. These results are similar to thoseobtained with aerobic oxidation of model compounds as discussed byFedorak, F. M. in “Geochemistry of Sulfur in Fossil Fuels,” ACSSymposium Series 429, American Chemical Society, Washington, D.C.,93-112, 1990. Additional reduction of thiophenic forms was found in thebiotreatment with BNL-TH-29 strain with variable levels of reduction insulfone, and sulfate forms and a slight increase in sulfoxides. Afterbiotreatment, there was a small increase of sulfide forms. The resultspresented in Table 5 indicate that thiophenic forms of sulfur present incoal have been reduced and altered by biotreatment. These chemicalchanges in the organic sulfur forms present in coals can occur only ifsulfur linkages are ruptured during biochemical treatment. These resultsare also consistent with those obtained by PY-GC-MS analysis asillustrated in FIG. 3.

Example 5 Trace Metal Analysis

50 mg samples of the desiccated coal obtained in Example 2 were digestedaccording to the nitric vapor ashing method of Thomas A. D., et al.,Talanta, 20, 469, 1973. The digested samples were dissolved in 1% HNO₃and analyzed by vapor gas inductively coupled plasma mass spectrometer(“ICP-MS”)model plasma Quad Plus 2. The results of the ICP-MS analysisare set forth in Table 6 below.

TABLE 6 Trace Metals Contents (μg/g) Trace Untreated Biotreated MetalsKentucky No. 8 Kentucky No. 8 V 157 99 Mn 168 41 Cu 143 0 Sr 1400 1040 Y80 57 Zr 148 88 La 83 53 Ce 148 140 Pb 76 1 Th 34 23 U 19 0

The results set forth in Table 6 indicate that during biochemicaltransformation of coals the depolymerization and desulfurization of coalis accompanied by a significant overall decrease in trace metals presentin coal.

The above experimental results obtained by two independent analyticalmethods, namely, PY-GC-MS and XANES, indicate that the biochemicaltreatment of coals according to the present invention provides a coalthat has been desulfurized, depolymerized and demineralized.

Thus, while there have been described what are presently believed to bethe preferred embodiments of the present invention, those skilled in theart will appreciate that other and further modifications can be madewithout departing from the true scope of the invention, and it isintended to include all such modifications and changes as come withinthe scope of the claims as appended herein.

What is claimed is:
 1. A method of preparing a mixture of modifiedbacterial strains selected through nutritional stress to upgrade a coalslurry which comprises: (i) providing at least a thermophilic bacterialstrain which grows on oil; (ii) nutritionally stressing saidthermophilic bacterial strain through sequential conditions comprisingdecreasing crude oil concentration and increasing coal concentrationuntil coal is essentially the sole carbon source and increasing salinityand trace metal concentration stepwise until said thermophilic bacterialstrain is capable of growing on coal as essentially the sole carbonsource at a salinity level between 1.5 weight % to 35 weight %, a tracemetal concentration from about 0.01 weight % to about 10 weight %, andpressure from about atmospheric to about 2500 psi; (iii) recovering saidnutritionally stressed bacterial strain; and (iv) forming a mixture ofsaid nutritionally stressed thermophilic bacterial strain with otherthermophilic bacterial strains which have been nutritionally stressedaccording to steps (ii) and (iii); (v) recovering said mixture ofnutritionally stressed thermophilic bacterial strains.
 2. The method ofclaim 1, wherein said nutritional stressing is conducted at atemperature greater than about 40° C.
 3. The method of claim 1, whereinsaid nutritionally stressed bacterial strain is selected from the groupconsisting of Achromobacter sp. BNL-4-23 (ATCC 55021), Sulfolobussolfataricus BNL-TH-29 (ATCC 55022), Sulfolobus solfataricus BNL-TH-31(ATCC 55023), Sulfolobus acidocaldarious BNL-TH-1 (ATCC 35091),Pseudomonas sp. BNL-4-24 (ATCC 55024), Leptospirillum ferrooxidansBNL-5-30 (ATCC 53992), Leptospirillum ferrooxidans BNL-5-31 (ATCC53993), Acinetobacter calcoaceticus BNL-4-21 (ATCC 53996), Arthrobactersp. BNL-4-22 (ATCC 53997), Acinetobacter calcoaceticus BNL-4-21s (ATCC55489), Arthrobacter sp. BNL-4-22s (ATCC 55490), Achromobacter sp.BNL-4-23s (ATCC 55491), Pseudomonas sp. BNL-4-24s (ATCC 55492), MixedCulture R.I.-1 (ATCC 55501), Leptospirillum ferrooxidans BNL-5-30s (ATCC55523), Leptospirillum ferrooxidans BNL-5-31s (ATCC 55524), Thiobacillusferrooxidans BNL-2-44s (ATCC 55525), Thiobacillus ferrooxidans BNL-2-45s(ATCC 55526), Thiobacillus ferrooxidans BNL-2-46s (ATCC 55527),Thiobacillus ferrooxidans BNL-2-47s (ATCC 55528), Thiobacillusferrooxidans BNL-2-48s (ATCC 55529), Thiobacillus ferrooxidans BNL-2-49s(ATCC 55530), Unknown BNL-NZ-3 (ATCC 55488), Mixed Culture R.I.-2 (ATCC55502), Mixed Culture R.I.-3 (ATCC 55503), Mixed Culture R.I.-4 (ATCC55504), Mixed Culture R.I.-5 (ATCC 55505), Mixed Culture R.I.-6 (ATCC55506), Mixed Culture R.I.-7 (ATCC 55507), Mixed Culture R.I.-8 (ATCC55508), Mixed Culture R.I.-9 (ATCC 55509), Mixed Culture R.I.-10 (ATCC55510), Mixed Culture R.I.-11(ATCC 55511), Mixed Culture R.I.-12 (ATCC55512), Mixed Culture R.I.-13 (ATCC 55513), Mixed Culture R.I.1-14 (ATCC55514), and mixtures thereof.
 4. The method of claim 1, wherein saidcoal slurry is lignite or bituminous coal.
 5. The method of claim 1,wherein said selection through nutritional stress is conducted at a pHrange from about 2 to about
 10. 6. The method of claim 5, wherein saidselection is conducted at a pH of
 4. 7. The method of claim 1, whereinsaid bacterial strain is modified to utilize coal as sole source ofcarbon at a condition selected from the group consisting of atemperature range from about 40° C. to about 85° C., pressure range fromatmospheric to about 2500 psi, a pH range from about 2 to about 10, asalinity range from about 1.5 weight % to about 35 weight % and a toxicmetal concentration from about 0.01 weight % to about 10 weight %.
 8. Amethod of preparing modified bacterial strains selected throughnutritional stress to upgrade a coal slurry of coal which comprises: (i)providing at least one thermophilic bacterial strain which grows on oil;(ii) nutritionally stressing said thermophilic bacterial strain throughsequential conditions comprising decreasing crude oil concentration andincreasing coal concentration until coal is essentially the sole carbonsource and increasing salinity and trace metal concentration stepwiseuntil said thermophilic bacterial strain is capable of growing on coalas essentially the sole carbon source at a salinity level between 1.5weight % to 35 weight %, a trace metal concentration from about 0.01weight % to about 10 weight %; and a pressure from about atmospheric toabout 2500 psi; and (iii) recovering said nutritionally stressedbacterial strain; wherein said at least one thermophilic bacterialstrain is selected from the group consisting of Achromobacter sp. BNL-4-23 (ATCC 55021), Sulfolobus solfataricus BNL-TH-29 (ATCC 55022),Sulfolobus solfataricus BNL-TH-31 (ATCC 55023), Sulfolobusacidocaldarious BNL-TH-1 (ATCC 35091), Pseudomonas sp. BNL-4-24 (ATCC55024), Leptospirillum ferrooxidans BNL-5-30 (ATCC 53992),Leptospirillum ferrooxidans BNL-5-31 (ATCC 53993), Acinetobactercalcoaceticus BNL-4-21 (ATCC 53996), Arthrobacter sp. BNL-4-22 (ATCC53997), Acinetobacter calcoaceticus BNL-4-21s (ATCC 55489), Arthrobactersp. BNL4-22s (ATCC 55490), Achromobacter sp. BNL-4-23s (ATCC 55491),Pseudomonas sp. BNL-4-24s (ATCC 55492), Mixed Culture R.I.-1 (ATCC55501), Leptospirillum ferrooxidans BNL-5-30s (ATCC 55523),Leptospirillum ferrooxidans BNL-5-31 s (ATCC 55524), Thiobacillusferrooxidans BNL-2-44s (ATCC 55525), Thiobacillus ferrooxidans BNL-2-45s(ATCC 55526), Thiobacillus ferrooxidans BNL-2-46s (ATCC 55527),Thiobacillus ferrooxidans BNL-2-47s (ATCC 55528), Thiobacillusferrooxidans BNL-2-48s (ATCC 55529), Thiobacillus ferrooxidans BNL-2-49s(ATCC 55530), Unknown BNL-NZ-3 (ATCC 55488), Mixed Culture R.I.-2 (ATCC55502), Mixed Culture R.I.-3 (ATCC 55503), Mixed Culture R.I.-4 (ATCC55504), Mixed Culture R.I.-5 (ATCC 55505), Mixed Culture R.I.-6 (ATCC55506), Mixed Culture R.I.-7 (ATCC 55507), Mixed Culture R.I.-8 (ATCC55508), Mixed Culture R.I.-9 (ATCC 55509), Mixed Culture R.I.-10 (ATCC55510), Mixed Culture R.I.-11 (ATCC 55511), (ATCC 55512), Mixed CultureR.I.-13 (ATCC 55513), Mixed Culture R.I.-14 (ATCC 55514), and mixturesthereof.